Figure 1. Inhibition of proteasome proteolytic activities sensitizes HeLa cells to ricin only after long incubations.

(A) HeLa cells were treated for 1, 2, 4, 6 or 8 h with graded doses of ricin in growth medium containing 1 μM Pi1 (●) or vehicle (DMSO, ○), and their subsequent ability to synthesize proteins was determined by measuring incorporation of [³⁵S]methionine into acid-precipitable material. Typical single assays are shown. (B) Top panel: cells were treated as described in (A), sensitivities to toxin (IC₅₀, toxin concentration required to reduce protein synthesis to 50% that of non-toxin treated controls) were determined, and fold protection (IC₅₀ Pi1-treated cells/IC₅₀ DMSO-treated cells) is displayed. Middle and bottom panels: cells were treated as described above, substituting the proteasome inhibitor ALLN (middle panel) or a mixture of the cathepsin inhibitors leupeptin and pepstatin (L+P, bottom panel) for Pi1, and substituting water for the vehicle for L+P treatment. Values are means±S.D. for three independent experiments. Broken line, no protective effect over that of treatment with vehicle only; n.d., not determined. (C) Cells were treated with a saturating dose of ricin for 4 h in the presence (+) or absence (−) of Pi1 (top panel), L+P (middle panel) or the secretion inhibitor BFA (bottom panel). Detergent-soluble extracts were separated by SDS/PAGE, and RTA (black arrowhead) and a proteolytically clipped RTA (white arrowhead) were revealed by immunoblotting. (D) HeLa cells were treated for 2, 4 or 6 h as described in (A), substituting the proteasome inhibitor cLβ-l for Pi1. Values are means±S.D. for three independent experiments. Broken line, no protective effect over that of treatment with DMSO only. (E) Cells were treated with a single low dose of ricin (1 ng/ml_, top panel) for increasing times in the presence of cLβ-l (●) or vehicle (DMSO, ○) and in parallel with cLβ-l (●) or vehicle (DMSO, ○) in the absence of ricin (bottom panel) and their subsequent ability to synthesize proteins was determined by measuring incorporation of [³⁵S]methionine into acid-precipitable material. Typical single assays are shown. Inset: toxin trafficking times, determined as in the top panel, in the presence of DMSO (D, ▼) or cLβ-l (c, ▽). Values are means±S.D. for three independent experiments.